PROJECT SUMMARY Glaucoma, a leading cause of blindness, is managed medically by treating the causal risk factor of increased intraocular pressure (IOP), which is typically observed prior to retina degeneration and loss of visual field. IOP is controlled in the anterior region of the eye, which contains the trabecular meshwork (TM) extracellular matrix, the anatomical pathway for drainage of aqueous humor fluid. Of the ~45 million cases of open angle glaucoma worldwide, ~3% are linked to mutations in myocilin, a protein highly expressed in the TM. Despite considerable research effort over ~20 years, little is known about the structure or function of myocilin. An improved molecular understanding of myocilin in its normal and disease states will change the paradigm for anti-glaucoma therapeutics by enabling agents that target the disease process instead of indirectly controlling IOP. Disease-associated mutations in myocilin are found throughout its sequence. In the prior grant period, we biophysically and structurally characterized the variants clustered in its C-terminal olfactomedin (mOLF) domain, lending critical new details and support for the predominant working hypothesis in which mutations localized to myoc-OLF lead to a gain of toxic function: Endoplasmic-reticulum (ER)-associated degradation is inhibited by an aberrant interaction between myocilin and the ER-resident chaperone Grp94, leading to amyloid deposits of mutant myocilin within TM cells, which are cytotoxic. The resulting accumulation of TM cell debris is thought to impede fluid outflow from the TM, causing IOP elevation. Continued structure/dysfunction studies of myocilin will not only contribute to our understanding of glaucoma and its role in the TM, but would also broaden our comprehension of the many other OLF domains, which are implicated broadly in physiology and diseases. The objectives of this proposal are to expand our molecular comprehension of structure and misfolding in myocilin-associated glaucoma as well as provide a path forward for functional studies and the discovery of small molecules that mitigate aberrant myocilin behavior. We will (1) elucidate the architecture of native full- length myocilin, which is dictated by N-terminal coiled-coils, and characterize biophysical and cellular properties of disease variants found therein, (2) clarify the interaction between myocilin and Grp94 at the molecular level, and (3) implement two high throughput assays. The expected outcomes are (1) the full scope of the misfolding disease mechanism for glaucoma-associated myocilin, (2) expansion of our knowledge of protein conformational disorders, (3) new insights into Grp94 chaperone biology, and (4) novel ligand assays based on the myoc-OLF structure and mOLF/Grp94 interaction for the identification of therapeutic small molecules.